PROJECT SUMMARY/ABSTRACT Acute Fatty Liver of Pregnancy (AFLP) is associated with the accumlation of 3-hydroxy fatty acids (3-HFAs), acute maternal liver disfunction, and significant maternal and perinatal mortality in the third trimester of pregnancy. Mothers with fetuses defective in the enzyme long chain 3-hydroxy acyl CoA dehydrogenase (LCHAD) develop AFLP. LCHAD deficiency leads to accumulation of 3-HFAs in the placenta that are shunted into maternal circulation damaging the maternal liver. The aim of the proposed research is to fill a gap in knowledge about whether 3-HFA is a causal factor in placenta and liver lipotoxicity observed with AFLP and, if it is, whether that information can be used in developing a therapy to treat 3-HFA-induced lipotoxicity. Preliminary data suggest that 3-HFAs exposure to hepatocyte cells induces lipoapoptosis and 3-HFAs exposure to placental trophoblasts induce necroptosis (i.e. inflammatory cell death). Initial studies indicate palmitoleate can protect against both 3-HFA-induced hepatocyte lipoapoptosis and trophoblast necroptosis in cell culture. The mechanism of 3 HFA-induced hepatocyte lipoapoptosis appears to involve: 1) pro-apoptotic FoxO3 nuclear activation; and 2) increased expression of FoxO3 downstream targets, like p53 upregulated modulator of apoptosis (PUMA). The central hypotheses are that: 1) toxic 3-HFAs accumulating during AFLP lead to placental trophoblast necroptosis and hepatocyte lipoapoptosis, and 2) dietary palmitoleate supplementation can mitigate 3-HFA-induced placental trophoblast and hepatocyte lipotoxicity and injury. The hypotheses will be tested through two specific aims (SAs): 1) elucidate the role of 3-HFA in lipotoxicity and its underlying mechanisms in of 3-HFA-induced lipotoxicity in the placenta and liver; and 2) mitigate 3-HFA-induced placental trophoblast and hepatocyte lipotoxicity with dietary palmitoleate supplementation. Hepatocytes and placental trophoblast cells will be treated in the presence or absence of 3-HFAs and examined for mitochondrial structure and function, pro- apoptotic mediators and biochemical markers of cell death and MAPK activation as a measure of lipotoxicity. The causal link for placenta and the liver lipotoxicity in AFLP with 3-HFA accumulation will be determined by injecting 3-HFAs in wild-type C57BL/6 pregnant mice to induce lipotoxicity and analyzing the markers of liver and placental lipotoxicity along with markers for cell death. Protection against trophoblast necroptosis will be studied via the activation of receptor interacting protein kinase 1, a key mediator of necroptosis, whereas protection against hepatocyte lipoapoptosis will be studied by investigating FoxO3 nuclear activation along with the Wnt3a-dependent cell survival pathways. The outcome of this project will contribute to Dr. Natajaran's long- term goal to develop foods high in palmitoleate as a therapeutic approach to mitigate placental and maternal liver injury in AFLP, which aligns with the Nebraska Center for the Prevention of Obesity through Dietary Molecules (NPOD)'s thematic focus of identifying biological, primarily food-borne signals that prevent, treat, and cure obesity-related diseases.